Comparative antisickling and antioxidant activities of Pseudobombax ellipticum cultivars in relation to their metabolite profiling using LC/MS

Pseudobombax ellipticum is native to South America and is cultivated worldwide mostly for its medicinal benefits. The plant is used traditionally in respiratory disorders such as dry cough, in the treatment of fever and stomach pain, and as an antimicrobial and analgesic. The antisickling and antioxidant effects of the flowers of P. ellipticum (Kunth) Dugand (red) and P. ellipticum cultivar alba (white) were compared using an in vitro assay in 2% sodium metabisulfite sickling induction model, DPPH, and metal chelation assays. Both red and white flowers exhibited antioxidant and antisickling activities. In DPPH assay, lower IC50 (34.89 ± 0.98 and 53.28 ± 1.14 μg mL−1) in red and white flowers respectively were detected relative to Trolox as a positive control (56.82 ± 0.87 μg mL−1). Comparable metal chelation activity (81.4 and 77.8 μM EDTA equivalent/mg) was detected in red and white flowers of both cultivars respectively. The average readings of the “reversal of sickling test “revealed a decrease in sickling percent from 49% to 15% in red flowers and to 18% in white flowers. Also, polymerization inhibition rate was increased from 0.34 to 1 and to 0.92 in red and white flowers respectively. Total phenolics, flavonoids and anthocyanins were quantified in red and white flowers as (163.9, 43.13 mg gallic acid equivalent/g extract), (71.92, 34.5 mg rutin equivalent/g extract) and (127.0, 85.9 mg pelargonidine-3-mono glucoside equivalent/kg extract), respectively. Liquid chromatography mass spectrometry (LC-MS) analysis was further employed for detection and identification of anthocyanins in flower extracts. Eight new anthocyanins were identified for the first time in genus Pseudobombax. These results reveal the potential role for both red and white flower extracts as possible antisickling agents in sickle cell anemia management.


Introduction
The family Bombacaceae is a small family of owering plants termed the Kapok family, referring to the cottony white bers (bombax or kapok) surrounding their seeds. 1,2 They are characterized by their unique brush-like owers in late winter. 1 Members of this family are used traditionally for the management of respiratory illnesses, gastrointestinal ailments and diabetes. 3,4 The genus Pseudobombax comprises 30 species; P. ellipticum (Kunth) Dugand also known as Bombax ellipticum is cultivated in Egypt. 5 Its owers show beautiful 5-inch long red (P. ellipticum (Kunth) Dugand) or white (P. ellipticum cultivar alba Hort.) stamens, looking very much like a shaving brush. 3 Few reports were found concerning the biological activities and chemical composition of P. ellipticum. Aqueous and ethanol extracts of the cortex exhibited signicant antioxidant activity. 6 From its stem bark, b-lupeol was isolated and showed potent gastro-protective effect. 7 Cyanidin-3,5-diglucoside was isolated from the owers. 2 Sickle cell anemia (SCA) is the most common genetic blood disorder worldwide. 8 According to estimates, Africa accounts for 75-85% of children born with SCA. Hemoglobin S (HbS) is produced instead of normal adult haemoglobin due to a mutation in the haemoglobin beta gene (HBB). 9 Normal RBCs have a 120 day lifespan and a exible biconcave disk-like shape that facilitates easy passage through the microvasculature 10 while under hypoxic conditions, HbS polymerizes, resulting in "sickle cells," hard and deformed RBCs with shortened life span (10-20 days) that cause decreased microcirculation and hemolysis. 11 Hence, antioxidants are very important in SCA management. 12,13 Pharmaceuticals that are now available for treating SCA are either not sufficiently effective, not entirely safe, or too expensive warranting for developing effective, safe, and affordable drugs from natural sources. 14 Interestingly, Ceiba pentandra (L.) Gaertn (Bombacaceae) is used traditionally in Congo to treat SCA and it's aqueous bark extract exhibited signicant antisickling and antithrombing activities. 15 Also, previous reports attributed the antisickling effect of several plants to their high anthocyanin content, present mainly in the owers. 16,17 As little data was available concerning P. ellipticum owers phytochemical makeup especially anthocyanins, and further prompted by the well reported anthocyanins antisickling potential, this study aims to identify phytoconstituents especially anthocyanins of potential effects in SCA using LC/MS. The antioxidant and antisickling activities of both P. ellipticum red owers (PR) as well as P. ellipticum cultivar alba white owers (PW) were compared in relation to their metabolite ngerprint.

Blood samples
The blood utilized in this study was drawn from patients known to have sickle cell anemia (Hemoglobin SS) from the hematology clinic at Cairo University Children Hospital "Abu L-Reesh Mounira". Informed consents were obtained from human participants of this study and all experiments were approved by the national ethic committee (No. MP3122) at Cairo University. Blood samples withdrawn from the selected patients were conrmed to contain homozygous (SS) red blood cells by haemoglobin electrophoresis carried out on cellulose acetate at pH 8.5. Samples were kept at 4°C once they were determined that they included SS red blood cells. 16 2.2. Plant material P. ellipticum red owers (PR) and P. ellipticum cultivar alba white owers.
(PW) were collected from Mostafa El Abed botanical garden, during May 2020. Dr Mohamed El Gibali, Senior Botanist & Consultant at Orman Botanic Garden, Giza, Egypt, kindly carried out the plant identication. A vouchered specimen of the plant, with serial number (20.09.2022) was kept at the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Preparation of ower extracts for antisickling assay
For preparation of extracts, fresh owers (50 g) from each plant were dissected by scissors to small pieces and separately extracted by 200 mL 100% methanol in 1 : 4 solid/solvent ratio using ultrasonic for 2 h. Extracts were then ltered via cotton pad and evaporated using rotavapor at temperature of 30°C that yielded 3 g residue for red owers and 2.8 g residue for white owers which were kept till further anaylsis. 18

Preparation of anthocyanins enriched extracts (AEE)
For another (50 g) of fresh owers, 200 mL acidied methanol (0.1% formic acid in methanol) was used in a ratio of 1 : 4 solid/ solvent and extraction was done in the dark, in amber colored bottles kept in the refrigerator to avoid against anthocyanins degradation. Extraction was done for 3 days followed by sonication for 10 minutes prior to ltration. Extract was evaporated using rotavapor at temperature of 30°C to yield 2.13 g residue for red owers and 1.75 g residue for white owers and kept at −20°C till further analysis. 18,19 2.5. Total phenolics assay Total phenolics were measured in ower samples using Folin-Cioacalteu assay. 20 A stock solution of 1 mg mL −1 of gallic acid in methanol was prepared as the standard, and seven serial dilutions were made at concentrations of 1000, 800, 600, 400, 200, 100, and 50 mg mL −1 . Samples from the AEE of white and red owers were prepared in a concentration of 4 mg mL −1 in methanol. Multimode plate reader (Fluostar Omega, BMG LABTECH, Germany) was used to record the outcomes. Six replicates of the 7 standards and 2 samples were pipetted into the wells of the plate measured at 630 nm. The average of the absorbance readings of the 6 replicates for gallic acid standard was recorded and calibration curve was established. From the established calibration curve, the linear regression equation was extracted for gallic acid standard (Fig. 1A). By substitution in the linear regression equation, the total phenolics were detected.

Total avonoids assay
Total avonoids were determined using colorimetric determination of avonoids -AlCl 3 complex. 21 A stock solution of 1 mg mL −1 of rutin in 100% methanol was prepared as the standard, and seven serial dilutions were made at concentrations of 1000, 600, 400, 200, 100, 50 and 10 mg mL −1 . Multimode plate reader (Fluostar Omega, BMG LABTECH, Germany) was used to record the outcomes. Six replicates of the 7 standards and 2 samples were pipetted into the wells of the plate measured at 420 nm. The average of the absorbance readings of the 6 replicates for rutin standard was recorded and calibration curve was established. From the established calibration curve, the linear regression equation was extracted for rutin standard (Fig. 1B). By substitution in the linear regression equation, the total avonoids were detected.

Total anthocyanins assay
Using potassium chloride (0.025 M, pH 1.0) and sodium acetate (0.4 M, pH 4.5) buffer systems, the total anthocyanins content was measured using the pH-differential method. 22 Each buffer solution (9 mL) was combined with 1 mL of AEE of red and white owers. The absorbance (Abs) was determined using UV/ Vis spectrophotometer, Jenway, England set at 520 and 700 nm, respectively.

Liquid chromatography-mass spectrometry (LC-MS) proling
An Agilent LC-MS system composed of an Agilent 1290 Innity II UHPLC coupled to an Agilent 6545 ESI-Q-TOF-MS in positive mode was used to obtain Ultra-high-performance liquid chromatograms. Aliquots (1 mL) of methanolic extracts (5 mg mL −1 in MeOH) were analyzed on a Kinetex phenyl-hexyl (1.7 mm, 2.1 × 50 mm) column. Isocratic elution of 90% A (A: 100% H 2 O + 0.1% formic acid) for 1 min followed by 6 min linear gradient elution to 100% B (95% MeCN + 5% H 2 O + 0.1% formic acid) with a ow rate of 0.4 mL min −1 . ESI conditions were set with the capillary temperature at 320°C, source voltage at 3.5 kV and a sheath gas ow rate of 11 L min −1 . Ions detected in the full scan at an intensity above 1000 counts at 6 scans per s, with an isolation width of 1.3 ∼ m/z, a maximum of 9 selected precursors per cycle and using ramped collision energy (5× m/z/100 + 10 eV). Purine C 5 H 4 N 4 [M + H] + ion (m/z 121.050873) and hexakis (1H,1H,3H-tetrauoropropoxy)-phosphazene C 18 H 18 F 24 N 3 O 6 P 3 [M + H] + ion (m/z 922.009798) were used as internal lock masses. 23 Metabolites were identied based on their retention times, molecular formulae and their fragmentation patterns, compared to earlier reported data aided with GNPS spectral library search and Sirius.
2.9. Antioxidant activity 2.9.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method. One of the most popular techniques for evaluating antioxidant activity is DPPH based in color change in DPPH solution to turn from deep violet color to light yellow color during the reaction. The EC50 of the compounds is used to display the assay's results. 24 In order to determine a range of concentrations within which the inhibitory concentration 50 (IC 50 ) lies, initial solutions of red and white owers AEE were prepared at concentration spanning from 1000 and 100 mg mL −1 in methanol. Extracts that exceeded 50% inhibition in any of the initial screening step, concentrations were serially diluted to provide 5 concentrations. Briey, 100 mL of freshly prepared DPPH reagent (0.1% in methanol) were added to 100 mL of the samples (dissolved in methanol) in 96 wells plate (n = 6), the reaction was incubated for 30 min in dark at room temperature. Decrease in DPPH colour intensity was assessed at 540 nm using a Multimode plate reader (Fluostar Omega, BMG LABTECH, Germany). Trolox was used as standard prepared at 100, 80, 60, 50, 40, 20 and 10 mg mL −1 . The results of six separate trials were combined to produce a mean and standard deviation (SD) for each sample. By converting the concentrations to their logarithmic value and choosing the nonlinear inhibitor regression equation log(inhibitor) vs. normalised response (variable slope equation), the data were analyzed using Microso Excel®, and the IC 50 value was determined using GraphPad Prism 5®. 25 The IC 50 was determined from preliminary screening to be less than 100 mg mL −1 . For each sample, the following serial dilutions were created: 100, 80, 60, 50, 40, 20 and 10 mg mL −1 .
2.9.2. Metal chelation assay. Metal chelation assay is based on that ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p0disulfonic acid monosodium salt hydrate) can bind to Fe 2+ ions to produce a blue color. The iron-ferrozine complex's blue color is attenuated upon binding of compounds and/or extracts to iron (Fe 2+ ). Using EDTA as a positive control, the colour reduction is used to gauge the compound's iron chelating action. 26 Serial dilutions (11) of 0.1 mM EDTA stock solution in water were created at concentrations of 5,10,15,20,25,30,35,40, 50, 60, and 70 mM. Samples of enriched extracts from red and white owers were prepared at a concentration of 0.625 mg mL −1 in methanol. The assay was conducted using the Santos et al. method, 26 with a few minor adjustments for microplates. In a 96well plate (n = 6), 20 mL of freshly made ferrous sulphate (0.3 mM) was combined with 50 mL of the samples. Then, each well received 30 mL of ferrozine (0.8 mM). For 10 minutes, the reaction mixture was incubated at room temperature. The reduction in the intensity of the generated colour was measured at 562 nm and percentage inhibition was calculated using this formula = (average absorbance of blank − average absorbance of the test/ average absorbance of blank) × 100. The multimode plate reader (Fluostar Omega, BMG LABTECH, Germany) was used to record the results. From the established calibration curve plotted from % inhibition of different concentrations of EDTA standard, the linear regression equation was extracted (Fig. 1C) and the results of the samples were presented as mM EDTA equivalent/mg sample. Aer subtraction from the blank (reagent + solvent), the average of the readings of the 6 replicates was taken.
2.10. Biological assay 2.10.1. Sickling reversal test. Using previously established methods, the plants' capacity to reverse the sickling status of the RBCs was determined. 16,27 Sickle cell blood was washed twice using ve times the volume of phosphate-buffered saline (PBS) with a pH of 7.4 by centrifugation at 4000 rpm for three minutes and the ower's total extracts were resuspended in absolute ethanol in a concentration of (0.25, 0.5 and 1 mg mL −1 ). For the control test, 50 mL of washed blood (WB) were added to 950 mL PBS into a clean Eppendorf tube and to 450 mL PBS and mixed with 500 mL of freshly prepared 2% sodium metabisulte into another clean one. For the inhibition test, 50 mL of WB were added to 400 mL PBS followed by 50 mL from the ower's total extracts and mixed with 500 mL of freshly prepared 2% sodium metabisulte into clean Eppendorf tubes. Control Tubes represent the initial percentage of sickling and sickling percentage aer induction with sodium metabisulte respectively. Ten mL from each mixture were spotted on a microscope slide covered with a cover slip. To seal the edges of the cover slip, paraffin wax was carefully applied and thus completely preventing air entry. By counting normal and sickled red cells under a 40× light microscope, the ability of the owers extracts to reverse the sickling of RBCs was observed and documented aer incubation of all slides for 2 h at 37°C. Morphologically, the red blood cells were classied as normal or sickled by observing their shapes. The elongated, C-like, or wrinkled shapes were considered sickled while biconcave or rounded shapes were taken to be normal. Using the following formula, the percentage sickled red blood cells was calculated: Red blood cells percent of sickling (%) = (number of counted sickled red blood cells divided by the total number of counted red blood cells) × 100. For each concentration, the experiment was conducted three times.

Polymerization inhibition test.
Following a previous procedure, the polymerization inhibition test was conducted. 11 In this procedure, the turbidity of the polymerizing solution of RBCs at a wavelength of 700 nm at a temperature of 26°C was measured. Aer adding 0.88 mL of freshly made 2% sodium metabisulte, into a clean cuvette, 0.1 mL of PBS and 0.02 mL of sickled blood, which had been washed twice in ve volumes of PBS (1 mL of blood in 5 mL of PBS) and then resuspended in PBS at a ratio of 1 : 1 was added. This serves as control test. For the inhibition test, 0.1 mL of PBS was substituted with 0.1 mL of the ower's total extracts that also were resuspended in absolute ethanol in a concentration of (0.25, 0.5 and 1 mg mL −1 ). For 60 minutes, the absorbance was measured at 700 nm immediately and at periodic intervals. Using the following formula, the rate of polymerization in percentage was computed: rate of polymerization (R p ) = [(nal absorbance − initial absorbance)/60] × 100. For each concentration, the experiment was repeated three times. At time zero (i.e., immediately aer addition of sodium metabisulte), the initial absorbance of the polymerizing solution of sodium metabisulte was measured and subtracted from the nal absorbance taken at the end of 60 min. Dividing the resulting value by 60 gives the rate of polymerization inhibition. The statistical signicance of difference was calculated by GraphPad Prism 9.5.1 using analysis of variance where value of p < 0.05 was considered signicantly different.

Total phenolics, avonoids and anthocyanins determination
Total phenolics expressed as gallic acid/equivalent were detected at 163.9 ± 6.05 and 43.13 ± 4.2 mg g −1 in red and white ower extracts, respectively. Likewise, higher avonoids level expressed as rutin/equivalent was detected in red owers at 71.92 ± 4.0 versus 34.5 ± 3.8 mg g −1 extract in white owers. Considering the abundance of anthocyanins in owers, their levels were estimated as mg pelargonidine-3-mono glucoside. The total anthocyanins content was detected at 127.0 and 85.9 mg kg −1 in red and white owers, respectively.

Antioxidant activities
Both red and white owers showed a potential DPPH radical scavenging activities with IC 50 value of (34.89 ± 0.98 and 53.28 ± 1.14 mg mL −1 ) respectively relative to Trolox with IC 50 (56.82 ± 0.87 mg mL −1 ). Red owers exhibited higher activity than white ones in case of DPPH assay. Likewise, red owers showed stronger metal chelation effect as another antioxidant action as manifested by 81.45 mM EDTA equivalent/mg sample versus 77.8 in case of white owers.

SCA in vitro assays
3.4.1. Sickling reversal test. Microscopical examination of sickle cells showed elongated or C-like shapes, whilst normal RBCs appeared rounded or biconcave ( Fig. 4 and 5). By using sodium metabisulte, sickled cell percentage increased from 8 to 49%. In contrast, red owers extracts (0.25, 0.5 and 1 mg mL −1 ) showed decrease in proportion of sickling to 20, 18 and 15% respectively, where white owers extracts showed 24,21 and 18% inhibition at a dose of 0.25, 0.5 and 1 mg mL −1 , respectively (Fig. 6).    ( Fig. 7). The rate of polymerization inhibition of the control was 0.34. Both assays revealed higher efficacy for red ower extract compared to white ower and are in agreement with antioxidant effect.

Discussion
SCA is a common blood disorder, affecting millions of people worldwide. 8 Ethno medicinal plants have a great importance in the treatment of various ailments including SCA where, phenolic compounds like avonoids and anthocyanins appeared to exert potential antisickling activity. 27,31,32 Anthocyanins especially exhibited promising antioxidant and antisickling activities. 16,17 Anthocyanins ability to attach to proteins may account for their antisickling effect and to further inhibition of the polymerization of deoxy HbS inside the RBCs. 16 Anthocyanins also exhibit a stabilizing effect on RBCs membrane and enhance the solubility of deoxy HbS, thereby inhibiting gel-ication. 16 In this study, the owers anthocyanins content was quantied and LC-MS revealed that nine anthocyanins were found enriched in both owers. Antioxidants are potential management strategies in SCA. 13,27 Interestingly, the owers extracts exhibited potential antioxidant activity by DPPH and metal chelation assays. Also, both ower extracts exhibited a potential antisickling and hemoglobin polymerization inhibition activities by preventing or reversing the sickling of RBCs. In accordance with previous reports higher content of active constituents mainly anthocyanins attributed to the higher  Sickling reversal activity of (PR) and (PW) at different extract concentrations. Fig. 7 Rate of polymerization inhibition (RPI%) of (PR) and (PW) at different extract concentrations with significant difference relative to control.
antioxidant and antisickling activities in PR. As illustrated, PR and PW were rich in phenolics, avonoids and anthocyanins with a higher content in case of PR bringing them (especially PR) as a promising trial in treatment of SCA.

Conclusion
The phytoconstituents of PR and PW especially anthocyanins were investigated using LC-MS. Their ethanolic extracts were able to stop or reverse sickling and haemoglobin polymerization of human sickled RBCs, thereby lowering the percentage of sickled cells and the rate of haemoglobin polymerization. Beside the antisickling activity, the extracts exhibited also potential antioxidant activity. These ndings may represent a primary screening step highlighting the antisickling potential of the owers of both cultivars. Further research is needed to detect the compounds responsible for the activity. Also, clinical studies need to be conducted to measure the activity in vivo and at the gene level.

Conflicts of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to inuence the work reported in this paper.